| Abstrakt: |
ABSTRACTDefinitive identification of the species in the Burkholderia cepaciacomplex by routine clinical microbiology methods is difficult. Phenotypic tests to identify B. multivoransandB. vietnamiensishave been established; more recent work indicates B. stabilismay also be identified by growth characteristics and biochemical tests. However, attempts to identify genomovars I and III have, thus far, proved unsuccessful. Previously, we demonstrated the utility of two primer pairs, directed to the rRNA operon, to specifically identify the B. cepaciacomplex in a PCR. One of these primer pairs, G1-G2, only amplified a DNA fragment from genomovars I and III and B. stabilisin a PCR with genomic DNA isolated from prototypical strains representing the five genomovars. Sequence analysis of the rRNA operon for all the genomovars indicated that this primer pair targeted a region shared by these isolates. Further analysis revealed a region of heterogeneity between genomovar III and B. stabilisinternal to the amplified product of G1-G2. Primers designed to target this region were tested with prototypical strains following an initial amplification with the G1-G2 primer pair. New primers specific for the prototypical genomovar III and B. stabiliswere designated SPR3 and SPR4, respectively. Analysis of 93 isolates representing 18 genomovar I, 13B. multivorans, 36 genomovar III, 11 B. stabilis, and 15 B. vietnamiensisisolates was performed. DNA from all isolates of genomovars I and III and B. stabiliswas amplified by G1-G2. Genomovar III isolates yielded a product with SPR3/G1 while B. stabilisamplified with SPR4-G1. Genomovar I isolates were amplified by either SPR3-G1 or SPR4-G1, but not both. B. multivoransyielded a product with SPR3-G1 but not G1-G2, and B. vietnamiensisisolates were negative in all PCRs. Thus using an algorithm with G1-G2, SPR3-G1, and SPR4-G1 primers in a PCR analysis, genomovar III isolates can be separated from B. stabilisand the identity of B. multivoransand B. vietnamiensiscan be confirmed. |
